Publications by authors named "Nijole Jasinskiene"

22 Publications

  • Page 1 of 1

Efficient population modification gene-drive rescue system in the malaria mosquito Anopheles stephensi.

Nat Commun 2020 11 3;11(1):5553. Epub 2020 Nov 3.

Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, 92697-3900, USA.

Cas9/gRNA-mediated gene-drive systems have advanced development of genetic technologies for controlling vector-borne pathogen transmission. These technologies include population suppression approaches, genetic analogs of insecticidal techniques that reduce the number of insect vectors, and population modification (replacement/alteration) approaches, which interfere with competence to transmit pathogens. Here, we develop a recoded gene-drive rescue system for population modification of the malaria vector, Anopheles stephensi, that relieves the load in females caused by integration of the drive into the kynurenine hydroxylase gene by rescuing its function. Non-functional resistant alleles are eliminated via a dominantly-acting maternal effect combined with slower-acting standard negative selection, and rare functional resistant alleles do not prevent drive invasion. Small cage trials show that single releases of gene-drive males robustly result in efficient population modification with ≥95% of mosquitoes carrying the drive within 5-11 generations over a range of initial release ratios.
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http://dx.doi.org/10.1038/s41467-020-19426-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7609566PMC
November 2020

Cas9-Mediated Gene-Editing in the Malaria Mosquito by ReMOT Control.

G3 (Bethesda) 2020 04 9;10(4):1353-1360. Epub 2020 Apr 9.

Department of Entomology, The Pennsylvania State University, University Park, PA 16802

Innovative tools are essential for advancing malaria control and depend on an understanding of molecular mechanisms governing transmission of malaria parasites by mosquitoes. CRISPR/Cas9-based gene disruption is a powerful method to uncover underlying biology of vector-pathogen interactions and can itself form the basis of mosquito control strategies. However, embryo injection methods used to genetically manipulate mosquitoes (especially ) are difficult and inefficient, particularly for non-specialist laboratories. Here, we adapted the ReMOT Control (ceptor-ediated vary ransduction of argo) technique to deliver Cas9 ribonucleoprotein complex to adult mosquito ovaries, generating targeted and heritable mutations in the malaria vector without injecting embryos. In , ReMOT Control gene editing was as efficient as standard embryo injections. The application of ReMOT Control to opens the power of CRISPR/Cas9 methods to malaria laboratories that lack the equipment or expertise to perform embryo injections and establishes the flexibility of ReMOT Control for diverse mosquito species.
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http://dx.doi.org/10.1534/g3.120.401133DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144067PMC
April 2020

Experimental population modification of the malaria vector mosquito, Anopheles stephensi.

PLoS Genet 2019 12 19;15(12):e1008440. Epub 2019 Dec 19.

Department of Microbiology & Molecular Genetics, University of California, Irvine, California, United States of America.

Small laboratory cage trials of non-drive and gene-drive strains of the Asian malaria vector mosquito, Anopheles stephensi, were used to investigate release ratios and other strain properties for their impact on transgene spread during simulated population modification. We evaluated the effects of transgenes on survival, male contributions to next-generation populations, female reproductive success and the impact of accumulation of gene drive-resistant genomic target sites resulting from nonhomologous end-joining (NHEJ) mutagenesis during Cas9, guide RNA-mediated cleavage. Experiments with a non-drive, autosomally-linked malaria-resistance gene cassette showed 'full introduction' (100% of the insects have at least one copy of the transgene) within 8 weeks (≤ 3 generations) following weekly releases of 10:1 transgenic:wild-type males in an overlapping generation trial design. Male release ratios of 1:1 resulted in cages where mosquitoes with at least one copy of the transgene fluctuated around 50%. In comparison, two of three cages in which the malaria-resistance genes were linked to a gene-drive system in an overlapping generation, single 1:1 release reached full introduction in 6-8 generations with a third cage at ~80% within the same time. Release ratios of 0.1:1 failed to establish the transgenes. A non-overlapping generation, single-release trial of the same gene-drive strain resulted in two of three cages reaching 100% introduction within 6-12 generations following a 1:1 transgenic:wild-type male release. Two of three cages with 0.33:1 transgenic:wild-type male single releases achieved full introduction in 13-16 generations. All populations exhibiting full introduction went extinct within three generations due to a significant load on females having disruptions of both copies of the target gene, kynurenine hydroxylase. While repeated releases of high-ratio (10:1) non-drive constructs could achieve full introduction, results from the 1:1 release ratios across all experimental designs favor the use of gene drive, both for efficiency and anticipated cost of the control programs.
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http://dx.doi.org/10.1371/journal.pgen.1008440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6922335PMC
December 2019

nanos-Driven expression of piggyBac transposase induces mobilization of a synthetic autonomous transposon in the malaria vector mosquito, Anopheles stephensi.

Insect Biochem Mol Biol 2017 08 1;87:81-89. Epub 2017 Jul 1.

Department of Molecular Biology and Biochemistry, University of California, Irvine, 3205 McGaugh Hall, Irvine, CA 92697-3900, United States; Department of Microbiology and Molecular Genetics, B240 Med Sci Bldg., School of Medicine, University of California, Irvine, CA 92697-4025, United States. Electronic address:

Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos. We assessed the feasibility of using the piggyBac transposon as a gene-drive mechanism to distribute anti-malarial transgenes in populations of the malaria vector, Anopheles stephensi. We designed synthetic gene constructs that express the piggyBac transposase in the female germline using the control DNA of the An. stephensi nanos orthologous gene linked to marker genes to monitor inheritance. Two remobilization events were observed with a frequency of one every 23 generations, a rate far below what would be useful to drive anti-pathogen transgenes into wild mosquito populations. We discuss the possibility of optimizing this system and the impetus to do so.
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http://dx.doi.org/10.1016/j.ibmb.2017.06.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580807PMC
August 2017

Highly efficient Cas9-mediated gene drive for population modification of the malaria vector mosquito Anopheles stephensi.

Proc Natl Acad Sci U S A 2015 Dec 23;112(49):E6736-43. Epub 2015 Nov 23.

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900; Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697-4500

Genetic engineering technologies can be used both to create transgenic mosquitoes carrying antipathogen effector genes targeting human malaria parasites and to generate gene-drive systems capable of introgressing the genes throughout wild vector populations. We developed a highly effective autonomous Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated protein 9 (Cas9)-mediated gene-drive system in the Asian malaria vector Anopheles stephensi, adapted from the mutagenic chain reaction (MCR). This specific system results in progeny of males and females derived from transgenic males exhibiting a high frequency of germ-line gene conversion consistent with homology-directed repair (HDR). This system copies an ∼ 17-kb construct from its site of insertion to its homologous chromosome in a faithful, site-specific manner. Dual anti-Plasmodium falciparum effector genes, a marker gene, and the autonomous gene-drive components are introgressed into ∼ 99.5% of the progeny following outcrosses of transgenic lines to wild-type mosquitoes. The effector genes remain transcriptionally inducible upon blood feeding. In contrast to the efficient conversion in individuals expressing Cas9 only in the germ line, males and females derived from transgenic females, which are expected to have drive component molecules in the egg, produce progeny with a high frequency of mutations in the targeted genome sequence, resulting in near-Mendelian inheritance ratios of the transgene. Such mutant alleles result presumably from nonhomologous end-joining (NHEJ) events before the segregation of somatic and germ-line lineages early in development. These data support the design of this system to be active strictly within the germ line. Strains based on this technology could sustain control and elimination as part of the malaria eradication agenda.
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http://dx.doi.org/10.1073/pnas.1521077112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4679060PMC
December 2015

Collagen-binding protein, Aegyptin, regulates probing time and blood feeding success in the dengue vector mosquito, Aedes aegypti.

Proc Natl Acad Sci U S A 2014 May 28;111(19):6946-51. Epub 2014 Apr 28.

Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, Rockville, MD 20852;

Mosquito salivary glands have important roles in blood feeding and pathogen transmission. However, the biological relevance of many salivary components has yet to be determined. Aegyptin, a secreted salivary protein from Aedes aegypti, binds collagen and inhibits platelet aggregation and adhesion. We used a transgenic approach to study the relevance of Aegyptin in mosquito blood feeding. Aedes aegypti manipulated genetically to express gene-specific inverted-repeat RNA sequences exhibited significant reductions in Aegyptin mRNA accumulation (85-87%) and protein levels (>80-fold) in female mosquito salivary glands. Transgenic mosquitoes had longer probing times (78-300 s, P < 0.0001) when feeding on mice compared with controls (15-56 s), feeding success was reduced, and those feeding took smaller blood meals. However, no differences in feeding success or blood meal size were found in membrane feeding experiments using defibrinated human blood. Salivary gland extracts from transgenic mosquitoes failed to inhibit collagen-induced platelet aggregation in vitro. Reductions of Aegyptin did not affect salivary ADP-induced platelet aggregation inhibition or disturb anticlotting activities. Our results demonstrate the relevance of Aegyptin for A. aegypti blood feeding, providing further support for the hypothesis that platelet aggregation inhibition is a vital salivary function in blood feeding arthropods. It has been suggested that the multiple mosquito salivary components mediating platelet aggregation (i.e., Aegyptin, apyrase, D7) represent functional redundancy. Our findings do not support this hypothesis; instead, they indicate that multiple salivary components work synergistically and are necessary to achieve maximum blood feeding efficiency.
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http://dx.doi.org/10.1073/pnas.1404179111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4024861PMC
May 2014

orco mutant mosquitoes lose strong preference for humans and are not repelled by volatile DEET.

Nature 2013 Jun 29;498(7455):487-91. Epub 2013 May 29.

Laboratory of Neurogenetics and Behavior, and Howard Hughes Medical Institute, The Rockefeller University, New York, New York 10065, USA.

Female mosquitoes of some species are generalists and will blood-feed on a variety of vertebrate hosts, whereas others display marked host preference. Anopheles gambiae and Aedes aegypti have evolved a strong preference for humans, making them dangerously efficient vectors of malaria and Dengue haemorrhagic fever. Specific host odours probably drive this strong preference because other attractive cues, including body heat and exhaled carbon dioxide (CO2), are common to all warm-blooded hosts. Insects sense odours via several chemosensory receptor families, including the odorant receptors (ORs), membrane proteins that form heteromeric odour-gated ion channels comprising a variable ligand-selective subunit and an obligate co-receptor called Orco (ref. 6). Here we use zinc-finger nucleases to generate targeted mutations in the orco gene of A. aegypti to examine the contribution of Orco and the odorant receptor pathway to mosquito host selection and sensitivity to the insect repellent DEET (N,N-diethyl-meta-toluamide). orco mutant olfactory sensory neurons have greatly reduced spontaneous activity and lack odour-evoked responses. Behaviourally, orco mutant mosquitoes have severely reduced attraction to honey, an odour cue related to floral nectar, and do not respond to human scent in the absence of CO2. However, in the presence of CO2, female orco mutant mosquitoes retain strong attraction to both human and animal hosts, but no longer strongly prefer humans. orco mutant females are attracted to human hosts even in the presence of DEET, but are repelled upon contact, indicating that olfactory- and contact-mediated effects of DEET are mechanistically distinct. We conclude that the odorant receptor pathway is crucial for an anthropophilic vector mosquito to discriminate human from non-human hosts and to be effectively repelled by volatile DEET.
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http://dx.doi.org/10.1038/nature12206DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3696029PMC
June 2013

Development of a population suppression strain of the human malaria vector mosquito, Anopheles stephensi.

Malar J 2013 Apr 26;12:142. Epub 2013 Apr 26.

Department of Molecular Biology & Biochemistry, University of California, Irvine, CA 92697-3900, USA.

Background: Transgenic mosquito strains are being developed to contribute to the control of dengue and malaria transmission. One approach uses genetic manipulation to confer conditional, female-specific dominant lethality phenotypes. Engineering of a female-specific flightless phenotype provides a sexing mechanism essential for male-only mosquito, release approaches that result in population suppression of target vector species.

Methods: An approach that uses a female-specific gene promoter and antibiotic-repressible lethal factor to produce a sex-specific flightless phenotype was adapted to the human malaria vector, Anopheles stephensi. Transposon- and site-specific recombination-mediated technologies were used to generate a number of transgenic An. stephensi lines that when combined through mating produced the phenotype of flight-inhibited females and flight-capable males.

Results: The data shown here demonstrate the successful engineering of a female-specific flightless phenotype in a malaria vector. The flightless phenotype was repressible by the addition of tetracycline to the larval diet. This conditional phenotype allows the rearing of the strains under routine laboratory conditions. The minimal level of tetracycline that rescues the flightless phenotype is higher than that found as an environmental contaminant in circumstances where there is intensive use of antibiotics.

Conclusions: These studies support the further development of flightless female technology for applications in malaria control programmes that target the vectors.
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http://dx.doi.org/10.1186/1475-2875-12-142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3648444PMC
April 2013

Exogenous gypsy insulator sequences modulate transgene expression in the malaria vector mosquito, Anopheles stephensi.

Proc Natl Acad Sci U S A 2013 Apr 12;110(18):7176-81. Epub 2013 Apr 12.

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA.

Malaria parasites are transmitted to humans by mosquitoes of the genus Anopheles, and these insects are the targets of innovative vector control programs. Proposed approaches include the use of genetic strategies based on transgenic mosquitoes to suppress or modify vector populations. Although substantial advances have been made in engineering resistant mosquito strains, limited efforts have been made in refining mosquito transgene expression, in particular attenuating the effects of insertions sites, which can result in variations in phenotypes and impacts on fitness due to the random integration of transposon constructs. A promising strategy to mitigate position effects is the identification of insulator or boundary DNA elements that could be used to isolate transgenes from the effects of their genomic environment. We applied quantitative approaches that show that exogenous insulator-like DNA derived from the Drosophila melanogaster gypsy retrotransposon can increase and stabilize transgene expression in transposon-mediated random insertions and recombinase-catalyzed, site-specific integrations in the malaria vector mosquito, Anopheles stephensi. These sequences can contribute to precise expression of transgenes in mosquitoes engineered for both basic and applied goals.
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http://dx.doi.org/10.1073/pnas.1304722110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3645527PMC
April 2013

Transgenic Anopheles stephensi coexpressing single-chain antibodies resist Plasmodium falciparum development.

Proc Natl Acad Sci U S A 2012 Jul 11;109(28):E1922-30. Epub 2012 Jun 11.

Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697-4500, USA.

Anopheles stephensi mosquitoes expressing m1C3, m4B7, or m2A10 single-chain antibodies (scFvs) have significantly lower levels of infection compared to controls when challenged with Plasmodium falciparum, a human malaria pathogen. These scFvs are derived from antibodies specific to a parasite chitinase, the 25 kDa protein and the circumsporozoite protein, respectively. Transgenes comprising m2A10 in combination with either m1C3 or m4B7 were inserted into previously-characterized mosquito chromosomal "docking" sites using site-specific recombination. Transgene expression was evaluated at four different genomic locations and a docking site that permitted tissue- and sex-specific expression was researched further. Fitness studies of docking site and dual scFv transgene strains detected only one significant fitness cost: adult docking-site males displayed a late-onset reduction in survival. The m4B7/m2A10 mosquitoes challenged with P. falciparum had few or no sporozoites, the parasite stage infective to humans, in three of four experiments. No sporozoites were detected in m1C3/m2A10 mosquitoes in challenge experiments when both genes were induced at developmentally relevant times. These studies support the conclusion that expression of a single copy of a dual scFv transgene can completely inhibit parasite development without imposing a fitness cost on the mosquito.
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http://dx.doi.org/10.1073/pnas.1207738109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3396534PMC
July 2012

Engineered resistance to Plasmodium falciparum development in transgenic Anopheles stephensi.

PLoS Pathog 2011 Apr 21;7(4):e1002017. Epub 2011 Apr 21.

Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, California, United States of America.

Transposon-mediated transformation was used to produce Anopheles stephensi that express single-chain antibodies (scFvs) designed to target the human malaria parasite, Plasmodium falciparum. The scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that inhibit either ookinete invasion of the midgut or sporozoite invasion of salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10, were fused to an Anopheles gambiae antimicrobial peptide, Cecropin A. Previously-characterized Anopheles cis-acting DNA regulatory elements were included in the transgenes to coordinate scFv production with parasite development. Gene amplification and immunoblot analyses showed promoter-specific increases in transgene expression in blood-fed females. Transgenic mosquito lines expressing each of the scFv genes had significantly lower infection levels than controls when challenged with P. falciparum.
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http://dx.doi.org/10.1371/journal.ppat.1002017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3080844PMC
April 2011

Female-specific flightless phenotype for mosquito control.

Proc Natl Acad Sci U S A 2010 Mar 22;107(10):4550-4. Epub 2010 Feb 22.

Oxitec Limited, 71 Milton Park, Oxford OX14 4RX, United Kingdom.

Dengue and dengue hemorrhagic fever are increasing public health problems with an estimated 50-100 million new infections each year. Aedes aegypti is the major vector of dengue viruses in its range and control of this mosquito would reduce significantly human morbidity and mortality. Present mosquito control methods are not sufficiently effective and new approaches are needed urgently. A "sterile-male-release" strategy based on the release of mosquitoes carrying a conditional dominant lethal gene is an attractive new control methodology. Transgenic strains of Aedes aegypti were engineered to have a repressible female-specific flightless phenotype using either two separate transgenes or a single transgene, based on the use of a female-specific indirect flight muscle promoter from the Aedes aegypti Actin-4 gene. These strains eliminate the need for sterilization by irradiation, permit male-only release ("genetic sexing"), and enable the release of eggs instead of adults. Furthermore, these strains are expected to facilitate area-wide control or elimination of dengue if adopted as part of an integrated pest management strategy.
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http://dx.doi.org/10.1073/pnas.1000251107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2826341PMC
March 2010

Microinjection of A. aegypti embryos to obtain transgenic mosquitoes.

J Vis Exp 2007 4(5):219. Epub 2007 Jul 4.

Department of Molecular Biology and Biochemistry, University of California, Irvine, USA.

In this video, Nijole Jasinskiene demonstrates the methodology employed to generate transgenic Aedes aegypti mosquitoes, which are vectors for dengue fever. The techniques for correctly preparing microinjection needles, desiccating embryos, and performing microinjection are demonstrated.
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http://dx.doi.org/10.3791/219DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2557089PMC
February 2009

The Anopheles gambiae vitellogenin gene (VGT2) promoter directs persistent accumulation of a reporter gene product in transgenic Anopheles stephensi following multiple bloodmeals.

Am J Trop Med Hyg 2007 Jun;76(6):1118-24

Department of Parasitology, School of Public Health and Tropical Medicine, Southern Medical University, Guang Zhou, GD, PR China.

Mosquitoes made resistant to pathogens through genetic engineering are proposed as a basis for developing a strategy to control disease transmission. Transgenic approaches that introduce exogenous antipathogen effector genes into mosquito genomes require cis-acting regulatory DNA to control tissue-, stage-, and sex-specific transgene expression. We show that control sequences derived from a vitellogenin-encoding gene of Anopheles gambiae, a major vector in sub-Saharan Africa, can direct expression of an exogenous gene in a tissue-, stage-, and sex-specific manner in Anopheles stephensi, a vector of urban malaria in southern Asia. Specific reporter gene expression was observed in fat-body tissues of transgenic blood-fed females, but not in transgenic males or non-blood-fed transgenic females. Multiple bloodmeals resulted in the continuous presence of reporter gene transcripts for at least 12 days. The persistent expression makes the heterologous promoter a good candidate for controlling transcription of engineered antipathogen effector genes in this important malaria vector.
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June 2007

Genetic control of malaria parasite transmission: threshold levels for infection in an avian model system.

Am J Trop Med Hyg 2007 Jun;76(6):1072-8

Department of Molecular Biology and Biochemistry, University of California, Irvine, California 92697-3900, USA.

Genetic strategies for controlling malaria transmission based on engineering pathogen resistance in Anopheles mosquitoes are being tested in a number of animal models. A key component is the effector molecule and the efficiency with which it reduces parasite transmission. Single-chain antibodies (scFvs) that bind the circumsporozoite protein of the avian parasite, Plasmodium gallinaceum, can reduce mean intensities of sporozoite infection of salivary glands by two to four orders of magnitude in transgenic Aedes aegypti. Significantly, mosquitoes with as few as 20 sporozoites in their salivary glands are infectious for a vertebrate host, Gallus gallus. Although scFvs hold promise as effector molecules, they will have to reduce mean intensities of infection to zero to prevent parasite transmission and disease. We conclude that similar endpoints must be reached with human pathogens if we are to expect an effect on disease transmission.
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June 2007

nanos gene control DNA mediates developmentally regulated transposition in the yellow fever mosquito Aedes aegypti.

Proc Natl Acad Sci U S A 2007 Jun 4;104(24):9970-5. Epub 2007 Jun 4.

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697, USA.

Transposable elements (TEs) are proposed as a basis for developing drive systems to spread pathogen resistance genes through vector mosquito populations. The use of transcriptional and translational control DNA elements from genes expressed specifically in the insect germ line to mediate transposition offers possibilities for mitigating some of the concerns about transgene behavior in the target vector species and eliminating effects on nontarget organisms. Here, we describe the successful use of the promoter and untranslated regions from the nanos (nos) orthologous gene of the yellow fever mosquito, Aedes aegypti, to control sex- and tissue-specific expression of exogenously derived mariner MosI transposase-encoding DNA. Transgenic mosquitoes expressed transposase mRNA in abundance near or equal to the endogenous nos transcript and exclusively in the female germ cells. In addition, MosI mRNA was deposited in developing oocytes and localized and maintained at the posterior pole during early embryonic development. Importantly, four of five transgenic lines examined were capable of mobilizing a second MosI transgene into the mosquito genome, indicating that functional transposase was being produced. Thus, the nos control sequences show promise as part of a TE-based gene drive system.
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http://dx.doi.org/10.1073/pnas.0701515104DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1891237PMC
June 2007

Functional characterization of the promoter of the vitellogenin gene, AsVg1, of the malaria vector, Anopheles stephensi.

Insect Biochem Mol Biol 2006 Sep 3;36(9):694-700. Epub 2006 Jun 3.

Department of Molecular Biology and Biochemistry, 3205 McGaugh Hall, University of California, Irvine, CA 92697 3900, USA.

Some genetic strategies for controlling transmission of mosquito-borne diseases call for the introgression of antipathogen effector genes into vector populations. Endogenous mosquito promoter and other cis-acting DNA sequences are needed to direct the expression of the effector molecules to maximize their efficacy. Vitellogenin (Vg)-encoding gene control sequences are candidates for driving tissue-, stage- and sex-specific expression of exogenous genes. One of the Anopheles stephensi Vg genes, AsVg1, was cloned and a full-length cDNA, as well as 850 base pairs adjacent to the 5'-end, were sequenced and characterized. Expression of AsVg1 is restricted to the fat body tissues of blood-fed females, and the amino acid sequence of the conceptual translation product is >85% identical to those of other anopheline Vgs. These characteristics support the conclusion that AsVg1 is a Vg-encoding gene. Functional analyses of the AsVg1 putative cis-regulatory sequences were performed using transgenic mosquitoes. The results showed that DNA fragments encompassing the 850 base pairs immediately adjacent to the 5'-end of the gene and the 3'-end untranslated region are sufficient to direct sex-, stage- and tissue-specific expression of a reporter gene. These data indicate that the AsVg1 promoter is a good candidate for controlling the expression of anti-pathogen effector molecules in this malaria vector mosquito.
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http://dx.doi.org/10.1016/j.ibmb.2006.05.011DOI Listing
September 2006

C1q and MBL, components of the innate immune system, influence monocyte cytokine expression.

J Leukoc Biol 2006 Jul 14;80(1):107-16. Epub 2006 Apr 14.

Department of Molecular Biology, University of California, Irvine, 92697, USA.

It has recently been recognized that the innate immune response, the powerful first response to infection, has significant influence in determining the nature of the subsequent adaptive immune response. C1q, mannose-binding lectin (MBL), and other members of the defense collagen family of proteins are pattern recognition molecules, able to enhance the phagocytosis of pathogens, cellular debris, and apoptotic cells in vitro and in vivo. Humans deficient in C1q inevitably develop a lupus-like autoimmune disorder, and studies in C1q knockout mice demonstrate a deficiency in the clearance of apoptotic cells with a propensity for autoimmune responses. The data presented here show that under conditions in which phagocytosis is enhanced, C1q and MBL modulate cytokine production at the mRNA and protein levels. Specifically, these recognition molecules of the innate immune system contribute signals to human peripheral blood mononuclear cells, leading to the suppression of lipopolysaccharide-induced proinflammatory cytokines, interleukin (IL)-1alpha and IL-1beta, and an increase in the secretion of cytokines IL-10, IL-1 receptor antagonist, monocyte chemoattractant protein-1, and IL-6. These data support the hypothesis that defense collagen-mediated suppression of a proinflammatory response may be an important step in the avoidance of autoimmunity during the clearance of apoptotic cells.
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http://dx.doi.org/10.1189/jlb.1105683DOI Listing
July 2006

Formation and loss of large, unstable tandem arrays of the piggyBac transposable element in the yellow fever mosquito, Aedes aegypti.

Transgenic Res 2004 Oct;13(5):411-25

Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA.

The Class II transposable element, piggyBac, was used to transform the yellow fever mosquito, Aedes aegypti. In two transformed lines only 15-30% of progeny inherited the transgene, with these individuals displaying mosaic expression of the EGFP marker gene. Southern analyses, gene amplification of genomic DNA, and plasmid rescue experiments provided evidence that these lines contained a high copy number of piggyBac transformation constructs and that much of this DNA consisted of both donor and helper plasmids. A detailed analysis of one line showed that the majority of piggyBac sequences were unit-length donor or helper plasmids arranged in a large tandem array that could be lost en masse in a single generation. Despite the presence of a transposase source and many intact donor elements, no conservative (cut and paste) transposition of piggyBac was observed in these lines. These results reveal one possible outcome of uncontrolled and/or unexpected recombination in this mosquito, and support the conclusion that further investigation is necessary before transposable elements such as piggyBac can be used as genetic drive mechanisms to move pathogen-resistance genes into mosquito populations.
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http://dx.doi.org/10.1007/s11248-004-6067-2DOI Listing
October 2004

High efficiency, site-specific excision of a marker gene by the phage P1 cre-loxP system in the yellow fever mosquito, Aedes aegypti.

Nucleic Acids Res 2003 Nov;31(22):e147

Department of Molecular Biology and Biochemistry, University of California, 3205 McGaugh Hall, Irvine, CA 92697-3900, USA.

The excision of specific DNA sequences from integrated transgenes in insects permits the dissection in situ of structural elements that may be important in controlling gene expression. Furthermore, manipulation of potential control elements in the context of a single integration site mitigates against insertion site influences of the surrounding genome. The cre-loxP site-specific recombination system has been used successfully to remove a marker gene from transgenic yellow fever mosquitoes, Aedes aegypti. A total of 33.3% of all fertile families resulting from excision protocols showed evidence of cre-loxP-mediated site-specific excision. Excision frequencies were as high as 99.4% within individual families. The cre recombinase was shown to precisely recognize loxP sites in the mosquito genome and catalyze excision. Similar experiments with the FLP/FRT site-specific recombination system failed to demonstrate excision of the marker gene from the mosquito chromosomes.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC275584PMC
http://dx.doi.org/10.1093/nar/gng148DOI Listing
November 2003

Gene vector and transposable element behavior in mosquitoes.

J Exp Biol 2003 Nov;206(Pt 21):3823-34

Center for Biosystems Research, University of Maryland Biotechnology Institute, College Park, MD 20742-4450, USA.

The development of efficient germ-line transformation technologies for mosquitoes has increased the ability of entomologists to find, isolate and analyze genes. The utility of the currently available systems will be determined by a number of factors including the behavior of the gene vectors during the initial integration event and their behavior after chromosomal integration. Post-integration behavior will determine whether the transposable elements being employed currently as primary gene vectors will be useful as gene-tagging and enhancer-trapping agents. The post-integration behavior of existing insect vectors has not been extensively examined. Mos1 is useful as a primary germ-line transformation vector in insects but is inefficiently remobilized in Drosophila melanogaster and Aedes aegypti. Hermes transforms D. melanogaster efficiently and can be remobilized in this species. This element is also useful for creating transgenic A. aegypti, but its mode of integration in mosquitoes results in the insertion of flanking plasmid DNA. Hermes can be remobilized in the soma of A. aegypti and transposes using a common cut-and-paste mechanism; however, the element does not remobilize in the germ line. piggyBac can be used to create transgenic mosquitoes and occasionally integrates using a mechanism other than a simple cut-and-paste mechanism. Preliminary data suggest that remobilization is infrequent. Minos also functions in mosquitoes and, like the other gene vectors, appears to remobilize inefficiently following integration. These results have implications for future gene vector development efforts and applications.
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http://dx.doi.org/10.1242/jeb.00638DOI Listing
November 2003

Development and applications of transgenesis in the yellow fever mosquito, Aedes aegypti.

Mol Biochem Parasitol 2002 Apr;121(1):1-10

Department of Molecular Biology and Biochemistry, University of California, 3205 McGaugh, Irvine, CA 92697-3900, USA.

Transgenesis technology has been developed for the yellow fever mosquito, Aedes aegypti. Successful integration of exogenous DNA into the germline of this mosquito has been achieved with the class II transposable elements, Hermes, mariner and piggyBac. A number of marker genes, including the cinnabar(+) gene of Drosophila melanogaster, and fluorescent protein genes, can be used to monitor the insertion of these elements. The availability of multiple elements and marker genes provides a powerful set of tools to investigate basic biological properties of this vector insect, as well as the materials for developing novel, genetics-based, control strategies for the transmission of disease.
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http://dx.doi.org/10.1016/s0166-6851(02)00028-2DOI Listing
April 2002
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